Abstract Colony Stimulating Factor-1 (CSF-1) is the primary regulator of the survival, proliferation and differentiation of mononuclear phagocytes, including tissue macrophages and osteoclasts. These cells play critical roles in the development and function of the tissues in which they reside. Not surprisingly, therefore, the similar phenotypes of CSF-1-deficient Csf1op/Csf1op and CSF-1R tyrosine kinase (c-Fms)-nullizygous (Csf1r-/-) mice are pleiotropic. However, the Csf1r-/- phenotype is more severe, indicating that some CSF-1R effects are mediated by CSF-1-independent CSF-1R activation. A new ligand for the CSF-1R, interleukin-34 (IL-34), has been identified. The major aim of this application is to define the biological role of IL-34 and establish the biological relationships between IL-34, CSF-1 and the CSF-1R in the regulation of hematopoietic differentiation to tissue macrophages and osteoclasts. The CSF-1R is expressed on primitive multipotent cells with characteristics of hematopoietic stem cells (HSC) and Csf1r-/- mice have reduced numbers of primitive hematopoietic cells. CSF-1 is either secreted as a glycoprotein (gp) or chondroitin sulfate-containing proteoglycan (pg), or expressed at the cell surface as a biologically active, membrane-spanning glycoprotein (cs). Mice exclusively expressing csCSF-1, or the precursors of gp, or pgCSF-1, in a normal tissue specific and developmental manner exhibit unique, but overlapping phenotypes that reflect specific roles of each isoform. Recent studies suggest that the csCSF and secreted CSF-1 have different actions on hematopoietic stem cells (HSC). Another aim of this application is to elucidate the roles of the different CSF-1 isoforms and of IL-34 in the regulation of HSC. Thus the overall aim is to define the biological roles of CSF-1 and IL-34 in the regulation of HSC and their differentiation to tissue macrophages and osteoclasts. The specific aims are to: 1. Determine the roles of the CSF-1R and its ligands in early hematopoiesis. 2. To define and compare the activities, expression patterns and regulation of IL-34 and CSF-1. 3. To establish the biological relationships between IL-34, CSF-1 and the CSF-1 receptor